Original Paper. Med Princ Pract 2015;24: DOI: /

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1 Original Paper Received: March 9, 2014 Accepted: November 17, 2014 Published online: January 23, 2015 Clinical Factors Associated with Negative Urinary Antigen Tests Implemented for the Diagnosis of Community-Acquired Pneumococcal Pneumonia in Adult Patients Hidehiro Watanabe a, b Tomonori Uruma a, b Gen Tazaki b Ryota Kikuchi a Takao Tsuji a Masayuki Itoh a a Department of Respiratory Medicine and Infection Control, Tokyo Medical University Ibaraki Medical Center, Inashiki, and b Department of Respiratory Medicine, Tokai University, Hachioji Hospital, Tokyo, Japan Key Words Urinary antigen test Community-acquired pneumonia Streptococcus pneumoniae Anticoagulation therapy Abstract Objective: This study investigated clinical factors associated with negative urinary antigen tests () implemented for the diagnosis of pneumococcal community-acquired pneumonia (CAP) in adult patients. Subjects and Methods: We reviewed the medical records of 755 adult patients who completed the in our hospital between 2009 and Of these, we evaluated 63 patients with bacteriologically confirmed definite pneumococcal CAP (33 were -positive, and 30 were -negative). Results: There was no significant difference between the -positive and the negative patients regarding age, dehydration, respiratory failure, orientation, blood pressure (ADROP) score (the CAP severity score proposed by the Japanese Respiratory Society), gender, white blood cell counts, liver/kidney function tests, or urinalysis. However, serum C-reactive protein (CRP) concentrations were 31% lower in the -negative patients than in the -positive patients (p = 0.02). Furthermore, the prothrombin time-international normalized ratio was 50% higher in the -negative patients than in the -positive patients, although the difference did not reach statistical significance (p = 0.06). The prevalence of comorbidities was similar in both -positive and -negative patients. However, warfarin had been prescribed in 8 (27%) of the -negative patients compared to only 1 (3%) of the -positive patients (odds ratio = 11.6; p = 0.01). Conclusions: These results suggested that low serum CRP concentrations and the use of warfarin increased the possibility with which false-negative results occurred in these patients with pneumococcal CAP S. Karger AG, Basel Introduction Community-acquired pneumonia (CAP) is highly prevalent and a major cause of death throughout the world. Streptococcus pneumoniae, the most common pathogenic microorganism, accounts for approximately 30% of the causative bacteria in CAP [1]. Due to the widespread use of rapid urine tests for the diagnosis of pneumococcal CAP and the publication of guidelines [2, 3] for CAP treatment with high-dose penicillin, the treatment karger@karger.com S. Karger AG, Basel /15/ $39.50/0 This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported license (CC BY-NC) ( applicable to the online version of the article only. Distribution permitted for non-commercial purposes only. Hidehiro Watanabe Department of Respiratory Medicine and Infection Control Tokyo Medical University Ibaraki Medical Center Chuou Ami, Inashiki (Japan) tokyo-med.ac.jp

2 outcomes for CAP has greatly improved. Because of its high sensitivity (77 87%) and specificity (97 100%) [1, 4], the Binax NOW (Alere, Waltham, Mass., USA) S. pneumoniae urinary antigen test (), a simple rapid immunochromatographic test that detects the C-polysaccharide antigen in urine, is widely used in the medical diagnosis of CAP [5]. Compared to conventional microbiologic tests, the Binax detected CAP in an additional 11.4% of patients [6]. CAP of initially unknown cause, as diagnosed with conventional microbiologic tests, has been attributable to S. pneumoniae in up to 57% of the cases after the introduction of the [7]. Hence, a number of studies and guidelines have suggested the usefulness of the as a powerful tool for the diagnosis of pneumococcal CAP [4]. In routine clinical practice, however, we often encounter puzzling cases of CAP where S. pneumoniae was ultimately judged to be a causative bacterium based on microbiologic examinations, while the result was negative [8]. The presence of such false-negative results imposes a significant limitation on the that compromises its clinical usefulness as a diagnostic tool for CAP [9]. However, the factors that affect the sensitivity of are largely unknown. In the present study, we reviewed the clinical records of patients with pneumococcal pneumonia to identify factors associated with decreased sensitivity of the. Subjects and Methods Study Population and Protocol This retrospective study included patients aged 18 years or older who had undergone the in the Emergency Room or at the Outpatient Clinic of the Tokai University Hachioji Hospital, a 500- bed city teaching hospital serving a population of approximately 560,000 in southwest Tokyo, Japan, from September 2009 to September This study was conducted in compliance with the requirements and under the authorization of the Tokai University Institutional Review Board (Certification No: 13R-158). The Binax NOW S. pneumoniae was performed in 755 patients with suspected pneumonia in the Emergency and Outpatient Departments. It was performed according to the instruction manual attached to the test kit. A total of 683 patients had negative, and 72 patients had positive results. Pneumococci were detected in sputum and/or other biological samples (see below) in 88 patients. A total of 63 of the 88 patients were analyzed, excluding 25 who did not have CAP. Microbiologic evaluation was performed as described previously [9]. The and various laboratory cultures were also performed simultaneously. Inclusion criteria for the assessment of pathogenic S. pneumoniae infection were: detection of Gram-positive diplococci in sputum samples containing 10 epithelial cells and 25 polymorphonuclear leukocytes per low-power field; isolation of 107 colony-forming units/ml S. pneumoniae in sputum culture, or the isolation of S. pneumoniae in any cultures of blood (two sets), pleural effusion or spinal fluid. Failure to satisfy the assessment criteria resulted in the exclusion from the study. According to the Japanese Respiratory Society s (JRS) diagnostic criteria [2], CAP was defined as an acute illness acquired in the community with the presence of new lung infiltration on a chest X-ray and with at least two of the following criteria: fever, cough, or purulent sputum. Diagnosis of definite pneumococcal CAP (DPnCAP) was made when patients satisfied both criteria for pathogenic S. pneumoniae infection and the criteria for CAP. Exclusion criteria were patients with any of the following: previously treated with antibiotics before admission, transferred to another hospital or received inpatient care at another hospital within 3 weeks, received pneumococcal vaccination within 5 days, had other acute conditions such as pulmonary edema, pulmonary embolism and malignancy during follow-up, had severe immunocompromised status with severe neutropenia [peripheral white blood cell (WBC) count <1,000/μl], postorgan/bone marrow transplant, or had a HIV infection. The susceptibility of penicillin to S. pneumoniae was determined using the Clinical and Laboratory Standards Institute (CLSI) penicillin breakpoints M100-S18 [10] (susceptible, 2 μg/ ml, penicillin-susceptible S. pneumoniae ; intermediate, 4 μg/ml, penicillin-intermediate S. pneumoniae ; resistant, 8 μg/ml, penicillin-resistant S. pneumoniae ). The urine tests were qualitative analyses and expressed a score of 0 3. The details of the standard score of the urine tests were as follows: urinary protein qualitative, 0: <30 mg/dl, 1: 30 to <100 mg/ dl, 2: 100 to <300 mg/dl, and 3: >300 mg/dl; urinary sugar qualitative, 0: <100 mg/dl, 1: 100 to <250 mg/dl, 2: 250 to <500 mg/dl, and 3: >500 mg/dl; urinary occult qualitative, 0: <0.06 mg/dl hemoglobin, 1: 0.06 to <0.135 mg/dl hemoglobin, 2: to <0.405mg/dl hemoglobin, and 3: >0.405 mg/dl hemoglobin. Statistical Considerations All statistical analyses were performed with Stat Mate IV (ATMS Co., Ltd., Tokyo, Japan). A descriptive analysis was performed for the demographic and clinical characteristics, and results are presented as means ± standard deviations for quantitative variables and numbers (percentages) for qualitative variables. For comparisons, the Mann-Whitney U test was performed for continuous variables which did not have parametric distributions, and Fisher s exact test was used for categorical variables. All p values were two-sided and considered statistically significant when they were Results The final analysis included 63 patients with DPnCAP, of whom 33 were -positive and 30 -negative. One of the 5 blood culture-positive cases had positive results in spinal fluid culture, and it was diagnosed as pneumococcal meningitis without CAP. Finally, this case was excluded from the CAP cases of this study ( table 1 ). 190 Watanabe /Uruma /Tazaki /Kikuchi /Tsuji / Itoh

3 Table 1. Details of the in 755 patients Total (n = 755) Positive (n = 72) Negative (n = 683) S N B P-E C-FS N B P-E C-F Culture S. pneumoniae detection Definite pneumococcal CAP S = Sputum; N = nasal secretion; B = blood; P-E = pleural effusion; C-F = cerebrospinal fluid. Resistant/Coexisting Bacteria The distribution of coexisting pathogens in patients with DPnCAP is shown in table 2. Of the 63 patients with DPnCAP, 17 (27.0%) had a polymicrobial infection; Staphylococcus aureus was detected in 5 patients, Moraxella catarrhalis in 4 patients, Klebsiella pneumoniae in 2 patients, and 7 patients had other infections. However, there was no significant difference in the distribution of coexisting pathogens between the -positive and the -negative patients with DPnCAP. Penicillin-resistant S. pneumoniae (at least penicillin intermediate resistant S. pneumoniae ) was isolated from 17 (27.0%) patients of the 63 patients with DpnCAP, without any significant difference noted between the -positive (9; 27.3%) and the -negative group (8; 26.7%). Sex, Age, Severities, and Laboratory Tests Of the 63 patients with DPnCAP, 42 (%) were male and 21 (%) were female ( table 3 ). The mean age was 64.7 years. There was no significant difference between the -positive and the -negative groups with respect to age, dehydration, respiratory failure, orientation, blood pressure (ADROP score; the CAP severity score proposed by the JRS), gender, blood WBC counts, liver/kidney function tests, or urinalysis ( table 3 ). However, the serum C-reactive protein (CRP) concentration was 31% lower in the -negative patients than in the -positive patients (p = 0.02). In contrast, the prothrombin time-international normalized ratio was 50% higher in the negative patients than in the -positive patients although the difference did not reach statistical significance (p = 0.06). Initial Antibiotics A penicillin/β-lactamase inhibitor combination was used in 22 (66.7%) of the 33 -positive patients (66.7%) and in 13 (43%) of the -negative patients (43%). The difference was not statistically different (p = Table 2. Distribution of mixed pathogens in patients with definite pneumococcal CAP stratified by the S. pneumoniae Definite pneumococcal CAP Total (n = 63) pos itive (n = 33) negative (n = 30) S. pneumoniae alone 4 (73.0) 25 (75.8) 21 (70.0) PSSP:PI-RSP 46:17 (27.0) 24:9 (27.3) 22:8 (26.7) Haemophilus influenzae Klebsiella pneumoniae Mycobacterium avium Moraxella catarrhalis Mycoplasma pneumoniae Pseudomonas aeruginosa Staphylococcus agalactiae Staphylococcus aureus Staphylococcus pyogenes Haemophilus influenzae + Staphylococcus aureus Influenza virus A Figures in parentheses are percentages. PSSP = Penicillin-susceptible S. pneumoniae; PI-RSP = penicillin-intermediate or -resistant S. pneumoniae. 0.08). Cephem antibiotics were used in 9 (27.3%) positive patients and in 12 (40.0%) -negative patients. Carbapenem antibiotics were used in 2 (6.1%) -positive patients and in 4 (13.3%) -negative patients. Underlying Diseases and Treatment Of the 63 patients, 51 (81%) with DPnCAP had at least one comorbidity ( table 4 ). The most common underlying disease was cardiovascular disease (11; 17.5%) and diabetes mellitus (11; 17.5%), followed by cerebrovascular disease (8; 12.5%), hypertension (8; 12.5%), liver disease (6; Clinical Factors Associated with Negative 191

4 Table 3. The comparison of background factors in positive and negative patients with definite pneumococcal CAP Definite pneumococcal CAP Total (n = 63) p value positive (n = 33) ne gative (n = 30) Gender, male/female 42/21 21/12 21/9 PSSP:PI-RSP 46:17 (27.0) 24:9 (27.3) 22:8 (26.7) Age, years 64.7 ± ± ± 19.3 ADROP score 1.0 ± ± ± WBC, 10 3 /μl 12,922 ± 5,540 13,003 ± 5,406 12,833 ± 5,775 CRP, mg/dl 16.8 ± ± ± * Cr, mg/dl 0.96 ± ± ± AST, IU/l 34.1 ± ± ± 28.3 ALT, IU/l 29.3 ± ± ± LDH, IU/l 253 ± ± ± INR 1.55 ± ± ± Urine-specific gravity ± ± ± Urine ph 6.14 ± ± ± 0.76 Urine protein qualitative, score 0.90 ± ± ± 0.86 Urine sugar qualitative, score 0.6 ± ± ± Urine occult qualitative, score 0.95 ± ± ± 1.27 Figures in parentheses are percentages. p > 0.2 shown with. PSSP = Penicillin-susceptible S. pneumoniae; PI-RSP = penicillin-intermediate or -resistant S. pneumoniae; Cr = creatinine; AST = aspartate aminotransferase; ALT = alanine aminotransferase; LDH = lactate dehydrogenase; INR = prothrombin time-international normalized ratio. * p < 0.05 for the comparison between -positive and negative patients. Table 4. Comorbidity characteristics of patients with definite pneumococcal CAP Definite pneumococcal CAP Total (n = 63) posi tive (n = 33) negative (n = 30) Underlying disease 51 (81) 27 (81.8) 24 (80) Bronchial asthma 4 (6.3) 2 (6.1) 2 (6.7) Cardiovascular disease 11 (17.5) 4 (12.1) 7 (23.3) Cerebrovascular disease 8 (12.7) 3 (9.1) 4 (13.3) Collagen disease 3 (4.8) 2 (6.1) 1 (3.3) COPD 4 (6.3) 3 (9.1) 1 (3.3) Diabetes mellitus 11 (17.5) 7 (21.2) 4 (13.3) Hypertension 8 (12.7) 4 (12.1) 4 (13.3) Liver disease 6 (9.5) 3 (9.1) 3 (10) Malignant disease 3 (4.8) 1 (3) 2 (6.7) Pulmonary fibrosis 4 (6.3) 3 (9.1) 1 (3.3) Renal disorder 2 (3.2) 1 (3) 1 (3.3) Other disease 5 (7.9) 2 (6.1) 3 (10) Figures in parentheses indicate percentages. Table 5. Treatment of underlying diseases Medicine positive (n = 33) Antihypertensive agents 4 5 Antiulcer agents 5 4 ICS + LABA 3 2 Oral corticosteroid 4 2 Antidiabetic agents 7 4 Anticoagulants 2 4 Warfarin* 1 8 Hepatoprotective agents 2 2 Others 2 1 No medication 6 6 negative (n = 30) ICS = Inhaled corticosteroid; LABA = long-acting β-agonist. * A significant p value (0.01) for the comparison between positive and negative patients. 192 Watanabe /Uruma /Tazaki /Kikuchi /Tsuji / Itoh

5 9.5%), bronchial asthma (4; 6.3%), COPD (4; 6.3%) and pulmonary fibrosis (4; 6.3%). There was no significant difference in the prevalence of comorbidities between the -positive and the -negative groups. However, warfarin had been prescribed in 8 (26.7%) -negative patients when compared to only 1 (3.0%) -positive patient (odds ratio = 11.6; p = 0.01), while the prescription of antiplatelets, antihypertensives, antidiabetics, and antiulcer drugs as well as of corticosteroids, bronchodilators, and hepatoprotective drugs were similar in the two groups of patients ( table 5 ). Discussion In this study, the false-negative was associated with lower serum CRP concentrations (p = 0.02) and a greater use rate of warfarin (p = 0.01). The prothrombin time-international normalized ratio was 50% higher in the -negative patients than in the -positive patients although the difference was not statistically significant (p = 0.06). In contrast, the positive or negative rate of was not influenced by age, sex, severity of pneumonia, the presence of penicillin-resistant S. pneumoniae or coexisting bacteria, nor was it influenced by WBC counts, or liver and kidney function tests. These findings suggest that an insufficient increase in CRP concentrations and the current use of warfarin reduced the sensitivity of the as a diagnostic tool for pneumococcal CAP. The clinical usefulness of the is often compromised by the false-negative result that confuses clinical decisions to select antibiotics. In our study, the false-negative rate of was 47.6% patients with DPnCAP, which is comparable to or somewhat higher than the previously reported value of 19 38% [8, 9, 11]. Bacterial colonization in the airway is known to be a possible cause of negative in patients with a positive sputum culture of S. pneumoniae [6]. To avoid a bias that may result from bacterial colonization, our study included only DPnCAP patients who satisfied the established diagnostic criteria for both CAP and pathologenic S. pneumoniae infection. In this study, two interesting features that were associated with the false-negative were identified. The first false-negative was associated with lower serum CRP concentrations when compared with positive cases. A probable explanation of this association was that a bacterial load might have been lower in the false-negative patients than in the positive patients. However, this explanation is not supported given that the severity of pneumonia, as determined by the ADROP score, was similar between the two groups of patients. An alternative explanation involved a possible interaction between C-polysaccharide in the pneumococcal capsule [pneumococcal C-polysaccharide (PnC)] and CRP. CRP had been shown to bind with phosphorylcholine residues in PnC, thereby serving as a carrier protein that allows the PnC to circulate in the blood and to be efficiently excreted in urine [12 14]. Because detects the PnC excreted in urine, if serum CRP levels are insufficiently increased, the amount of PnC excreted in urine would be decreased, potentially leading to the false-negative. The second false-negative was also associated with the current use of warfarin. DPnCAP patients treated with warfarin for underlying cerebro- and cardiovascular diseases were 11.6 times more likely to have an opportunity to yield a negative result. In contrast, the positive or negative rate of was not influenced by the prevalence of cerebro- and cardiovascular diseases per se, or by the use of antiplatelets and other medications ( table 5 ). These findings suggest that the use of warfarin may mechanistically be linked to the false-negative result of. However, the reason for the association between the warfarin usage and the negative is currently unclear. We speculate that as one probability, warfarin might have controlled the binding of PnC and CRP through anticoagulation factors and ionic channels thereby leading to both insufficient CRP concentration and internal warfarin that probably produced the falsenegative. The present study has several limitations. First, this is a retrospective study, so some of the sampling biases could have confounded the results. Second, the was implemented only once when patients visited the emergency room or the outpatient clinic. Later timing of the urine sampling (for example, on the day after admission) could have increased the sensitivity of the. Three cases which had positive blood cultures were -negative in this study. Two of 3 cases which were -negative had received anticoagulation therapy (one of them had taken warfarin). Furthermore, the CRP concentrations of these 2 cases were lower than that of positive cases. In the last case, which was -negative, there was no influence of medicine, and a high concentration of CRP was observed (25.7 mg/dl). In this case, no reason for the negative results could be found. As described above, the may have shown positive results when checking on the following day. Finally, as for the selection of the initial antibacterial agent, there was no significant difference to penicillin se- Clinical Factors Associated with Negative 193

6 lection of initial antibacterial agent between the positive group and the negative group (p = 0.08). However, the negative patients who were diagnosed with the complication of atypical pneumonia were treated with the combined therapy of β-lactam antibacterial agent and macrolide. The information of negative might have affected the decision of clinicians to diagnose the complication of atypical pneumonia. In the region where the ratio of the atypical pneumonia especially in CAP is high [15], these results suggested that information from later is valuable for clinical decision-making because empiric antibiotic prescriptions would have already been started. Conclusions The present study suggested that an insufficient increase in CRP concentrations and the current use of warfarin are factors associated with negative results implemented for the diagnosis of CAP in adult patients. While further research needs to be conducted to confirm these results, physicians should make a clinical diagnosis of pneumococcal or nonpneumococcal pneumonia while keeping in mind that patients with relatively low CRP values and those receiving warfarin are prone to be associated with the false-negative results. Disclosure Statement The authors declare that there is no conflict of interest. References 1 Said MA, Johnson HL, Nonyane BA, et al: Estimating the burden of pneumococcal pneumonia among adults: a systematic review and meta-analysis of diagnostic techniques. PLoS One 2013; 8:e Kohno S, Seki M, Watanabe A; CAP Study Group: Evaluation of an assessment system for the Japanese Respiratory Society 2005: A-DROP for the management of CAP in adults. Intern Med 2011; 50: Mandell LA, Wunderink RG, Anzueto A, et al; Infectious Diseases Society of America; American Thoracic Society: Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults. Clin Infect Dis 2007; 44(suppl 2):S27 S72. 4 Smith MD, Sheppard CL, Hogan A, et al; South West Pneumococcus Study Group: Diagnosis of Streptococcus pneumoniae infections in adults with bacteremia and community-acquired pneumonia: clinical comparison of pneumococcal PCR and urinary antigen detection. J Clin Microbiol 2009; 47: Ishida T, Hashimoto T, Arita M, et al: A 3-year prospective study of a urinary antigendetection test for Streptococcus pneumoniae in community-acquired pneumonia: utility and clinical impact on the reported etiology. J Infect Chemother 2004; 10: Kobashi Y, Yoshida K, Miyashita N, et al: Evaluating the use of a Streptococcus pneumoniae urinary antigen detection kit for the management of community-acquired pneumonia in Japan. Respiration 2007; 74: van Mens SP, Meijvis SC, Endeman H, et al: Longitudinal analysis of pneumococcal antibodies during community-acquired pneumonia reveals a much higher involvement of Streptococcus pneumoniae than estimated by conventional methods alone. Clin Vaccine Immunol 2011; 18: Piso RJ, Iven-Koller D, Koller MT, et al: The routine use of urinary pneumococcal antigen test in hospitalised patients with community acquired pneumonia has limited impact for adjustment of antibiotic treatment. Swiss Med Wkly 2012; 142:w Briones ML, Blanquer J, Ferrando D, et al: Assessment of analysis of urinary pneumococcal antigen by immunochromatography for etiologic diagnosis of community-acquired pneumonia in adults. Clin Vaccine Immunol 2006; 13: Clinical and Laboratory Standards Institute: Performance Standards for Antimicrobial Susceptibility Testing: Eighteenth Informational Supplement M100-S18. Wayne, CLSI, Sordé R, Falcó V, Lowak M, et al: Current and potential usefulness of pneumococcal urinary antigen detection in hospitalized patients with community-acquired pneumonia to guide antimicrobial therapy. Arch Intern Med 2011; 171: Abernethy TJ, Avery OT: The occurrence during acute infections of a protein not normally present in the blood. 1. Distribution of the reactive protein in patients sera and the effect of calcium on the flocculation reaction with C polysaccharide of pneumococcus. J Exp Med 1941; 73: Kaplan MH, Volanakis JE: Interaction of C- reactive protein complexes with the complement system. 1. Consumption of human complement associated with the reaction of C- reactive protein with pneumococcal C-polysaccharide and with the choline phosphatides, lecithin and sphingomyelin. J Immunol 1974; 112: Holzer TJ, Edwards KM, Gewurz H, et al: Binding of C-reactive protein to the pneumococcal capsule or cell wall results in differential localization of C3 and stimulation of phagocytosis. J Immunol 1984; 133: Behbehani N, Mahmood A, Mokaddas EM, et al: Significance of atypical pathogens among community-acquired pneumonia adult patients admitted to hospital in Kuwait. Med Princ Pract 2005; 14: Watanabe /Uruma /Tazaki /Kikuchi /Tsuji / Itoh